MOCK validation on Fungal ITS analysis tutorial

[Manuss_Ponce (Manuel Ponce Alonso)]

Hi,
I’m following the MOCK validation section from the Fungal ITS analysis tutorial. I’m working with a set of ITS2 reads from Illumina Miseq run, and when I tried to validate my pipeline with the mock community used in that tutorial (mock-25), I found that my pipeline didn´t find any ITS2 sequence. Is there any mock community specific to ITS2 region?

My pipeline is based on that plugin: Q2-ITSxpress: A tutorial on a QIIME 2 plugin to trim ITS sequences and the error is found in that step:

qiime itsxpress trim-pair-output-unmerged --i-per-sample-sequences MOCK/UPSTREAM/paired-end-demux.qza --p-region **ITS2** --p-taxa F --p-threads 8 --o-trimmed MOCK/UPSTREAM/trimmed2.qza

Thanks a lot

[Nicholas_Bokulich]

Hi @Manuss_Ponce,
The issue is that itsxpress expects the reads to be in forward orientation; those mock reads are actually in reverse orientation for some reason.

I think you may be able to just switch the input fastq files, i.e., import the forward read as “reverse” and the reverse read as “forward”. Let me know if that works — it would be an easy workaround for that tutorial!

If that does not work, you will need to reverse complement the reads on input.

Good luck!

cc: @Adam_Rivers

[Adam_Rivers]

That’s correct, ITSxpress does not currently support primer sets in the reverse orientation. I was planning to add that featute, but the US federal government shutdown means I can’t. Most primer ITS1 and ITS2 primer sets are not reversed though so it should work for most people. The dataset in the tutorial works fine.

[Manuss_Ponce (Manuel Ponce Alonso)]

OK, thanks a lot for the clarification! I’ll try your instructions and post if it works!

Regards